LOCUS       sequence-378559-        7036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RvCAHS3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7036)
  AUTHORS   Tanaka A, Nakano T, Watanabe K, Masuda K, Honda G, Kamata S, Yasui 
            R, Kozuka-Hata H, Watanabe C, Chinen T, Kitagawa D, Sawai S, Oyama 
            M, Yanagisawa M, Kunieda T
  TITLE     Stress-dependent cell stiffening by tardigrade tolerance proteins 
            that reversibly form a filamentous network and gel.
  JOURNAL   PLoS Biol. 2022 Sep 6;20(9):e3001780. doi: 
            10.1371/journal.pbio.3001780. eCollection 2022 Sep.
  PUBMED    36067153
REFERENCE   2  (bases 1 to 7036)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Biol."; date: "2022-09-6"; volume: "20(9):e3001780. doi"; pages: " 
            10.1371/journal.pbio.3001780. eCollection 2022 Sep"
COMMENT     Sequence Label: pEThT-RvCAHS3
FEATURES             Location/Qualifiers
     source          1..7036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             984..1001
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1002..1022
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1023..1676
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1683..1700
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1753..1771)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1767..1814
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1851..2306
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1938..1957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2148..2169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2332..2436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2437..2505,2506..3297)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2655..2674)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3468..4056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3957..3976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4210..4227
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4242..4381
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4307..4326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4467..4489
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4483..4674)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5483..6565)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6527..6546
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6566..6643)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6849..6868
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6952..6971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6952..6970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6971..6995
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             7010..7032
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgcacc atcaccatca ccatggatca atgtcttccc gacagaacca gcaatcgtcc
       61 agccaacact cgtcctccag ccagcaaggt ggtcaaggtg gtcaaggtgt tcaaggaagt
      121 tccagctact cgcgcaccga ggtccacacc agcagtggag gacctaccat cggtggagcc
      181 cagcgaactg tccccgtccc ccctggatct cactccgagg tccatgagga gcgtgaggtc
      241 atcaagcatg gtaccaaaac cgaaagcgag acccacgtcg tcaccgtccc agtgacaact
      301 ttcggcagca ccaacatgga atctgtccga accggcttca ccgtcaccca agacaagaac
      361 ttgaccgttg ctgctcccaa catcgctgct cccatccaca gcaacctcga ccttaacctc
      421 ggcggtggag ctcgcgctga aattaccgca gggaccaccg ttgacttgag caagatccag
      481 cgcaaggatt tgggacctga agagtatgct cgctacaagg ccaaggtcga gcaactggcc
      541 aggcaagatg agcaagacgc gggtatgcgc gctgcccagt accgagagga agtggagcgt
      601 gatgccgaac tcatccgaca gatcttggag cgacaacaca tccgtgatct tgaattccgc
      661 aaggaaatgg ttgagaacca agtcaaccga caagagagag aaatccagct ggaggctgag
      721 tacgcaatgc gagccctcga gcttgagcgc aatgccgcta aagaggcttt ggaaagcgcc
      781 aaggcccaga ctaacgtcaa tgtcaaggtc gagtccgcta ttggtaccac cgtctccaag
      841 ggtgcaatcc agacttccgc cgacaagagc agcaccacca agactggacc caccaccgtc
      901 actcagatta aacataccga acaacacact gaacgccgat agaattcgat atcaagctta
      961 tcgatgctag cactagtcct aggctggttc cgcgtgggtc cgagaacctg tacttccagg
     1021 gctcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
     1081 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
     1141 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
     1201 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
     1261 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
     1321 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
     1381 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
     1441 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
     1501 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
     1561 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
     1621 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaataactcg
     1681 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     1741 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1801 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     1861 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1921 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1981 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2041 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2101 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2161 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2221 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2281 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2341 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2401 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2461 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2521 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2581 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2641 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     2701 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     2761 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     2821 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     2881 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     2941 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3001 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3061 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3121 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3181 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3241 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3301 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3361 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3421 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3481 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3541 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3601 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3661 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3721 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3781 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3841 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3901 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3961 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4021 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4081 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4141 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4201 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4261 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4321 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4381 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4441 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4501 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4561 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4621 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4681 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4741 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4801 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4861 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4921 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4981 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5041 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5101 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5161 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5221 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5281 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5341 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5401 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5461 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5521 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5581 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5641 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5701 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5761 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5821 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5881 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5941 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6001 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6061 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6121 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6181 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6241 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6301 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6361 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6421 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6481 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6541 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6601 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6661 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6721 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6781 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6841 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6901 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     6961 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     7021 ttaagaagga gatata
//
